2-hydroxy-3-phenoxypropyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of 2-Hydroxy-3-phenoxypropyl acrylate was evaluated in the three in vitro studies required by REACh Regulation (OECD 471, OECD 476, OECD 487).

All the three studies showed negative results indicating that 2-Hydroxy-3-phenoxypropyl acrylate is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2016 - 13 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

A technical error was noted in the first main experiment: the sampling volume of the test item was not taken into account in the calculation of the volume of vehicle to add for the preparation of stock formulation. However, if the density measured in the preliminary test is extrapolated, the volume of test item sampled for the first main experiment was 20 µL. Thus, the corrected concentration of the stock formulation was 15.79 mg/mL (instead of 16.01 mg/mL), corresponding to a difference of -1.37%. Since the difference is < ±10% (criterion based on the formulation galenic, i.e. a solution), this minor deviation was considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. The historical data used for the validation of long treatment period without S9 mix (24 hours treatment + 0 hour recovery) were generated with non-audited data from non-GLP studies. These data were performed in compliance with CiToxLAB France’s standard operating procedures. Since CiToxLAB France is a Test facility certified by the French National Authorities for Good Laboratory Practice, and the procedures undertaken are the same, this deviation is considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. Moreover, the corresponding mean frequency of micronucleated cells in the vehicle control was 3‰ in the first experiment or 2‰ in the second experiment, therefore = 5‰ as specified in the acceptance criteria.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10%(v/v) heat inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).

The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:
- 1.25, 2.5, 5, 10, 20, 30, 40 and 80 µg/mL, for the 3-hour treatment in the first experiment,
- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL, for the 24-hour treatment in the first experiment,
- 1.25, 2.5, 5, 6.25, 7.5, 8.75, 10 and 15 µg/mL, both for the 3- and 24-hour treatments in the second experiment.

Experiments with S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:
- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL in the first experiment,
- 7.5, 15, 30, 40, 45, 50, 55 and 60 µg/mL in the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: the test item was found to be soluble in DMSO and the highest recommended dose-level could be reached using a solution of the test item at a concentration of 400 mg/mL and a treatment volume of 0.5% (v/v) in culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

For the preliminary cytotoxicity test and both cytogenetic experiments:
Without S9 mix: 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix: 3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells / dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

no

Key result

Species / strain:

other: mouse lymphoma L5178Y TK+/- cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Emulsion: >= 1000 µg/mL at the end of the short treatment period; no emulsion at the end of the long treatment period
- Definition of acceptable cells for analysis: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. 1995, according to the following criteria:
* micronuclei should be clearly surrounded by a nuclear membrane,
* the micronucleus area should be less than one-third of the area of the main nucleus,
* non-refractility of the micronuclei,
* micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
* micronuclei should be located within the cytoplasma of the cell,
* only mononucleated cells with a number of micronuclei <= 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Using a solution of the test item in the vehicle at a concentration of 400 mg/mL and a treatment volume of 0.5 % (v/v) in culture medium, the highest recommended dose-level of 2000 µg/mL was achievable. Thus, the dose levels selected for the treatment of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the highest dose-level of 2000 µg/mL, the pH of the culture medium was approximately 7.7 (as for the vehicle control) and the osmolality was 350 mOsm/kg H2O (394 mOsm/kg for the vehicle control). Therefore, none of the selected dose-levels was considered to produce extreme culture conditions and the highest recommended dose-level of 2000 µg/mL could be selected as the highest dose-level for the main experiments.

An emulsion was observed in the culture medium at the dose-levels = 1000 µg/mL, at the end of the short treatment period. No emulsion was observed in the culture medium at any of the tested dose-levels at the end of the long treatment period.

Following the 3-hour treatments with and without S9 mix, a slight to severe cytotoxicity was observed from the lowest tested dose-level (i.e. 4 µg/mL), as shown by a 28 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a moderate to severe cytotoxicity was observed at dose levels = 40 µg/mL as shown by a 48 to 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached document.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document.
- Negative (solvent/vehicle) historical control data: see attached document.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: population doubling.

Conclusions:

2-Hydroxy-3-phenoxypropyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
 

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK^+/-.

 

After a preliminary toxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments with (3h treatment) and without (3hr and 24h treatment) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

 

The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Experiments without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:

- 1.25, 2.5, 5, 10, 20, 30, 40 and 80 µg/mL, for the 3-hour treatment in the first experiment,

- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL, for the 24-hour treatment in the first experiment,

- 1.25, 2.5, 5, 6.25, 7.5, 8.75, 10 and 15 µg/mL, both for the 3- and 24-hour treatments in the second experiment.

 

No emulsion was observed in the culture medium at any dose-levels, at the end of the treatment periods.

 

Following the 3-hour treatments, a cytotoxicity was induced at dose-levels = 10 µg/mL in the first experiment, and at dose-levels = 5 µg/mL in the second experiment, as shown by a 30 to 100% decrease in the PD.

Following the 24-hour treatments, a cytotoxicity was induced at dose-levels = 10 µg/mL in the first experiment, and at dose-levels = 8.75 µg/mL in the second experiment, as shown by a 29 to 100% decrease in the PD.

 

The dose-levels selected for micronucleus analysis were as follows:

- 1.25, 2.5 and 5 µg/mL for the 3-hour treatment in the first experiment, the latter inducing no decrease in the PD but higher dose-levels being too cytotoxic,

- 2.5, 5 and 6.25 µg/mL for the 3-hour treatment in the second experiment, the latter inducing a 46% decrease in the PD and higher dose-levels being too cytotoxic,

- 1.25, 2.5 and 5 µg/mL for the 24-hour treatment in the first experiment, the latter inducing only a 7% decrease in the PD but higher dose-levels being too cytotoxic,

- 7.5, 8.75 and 10 µg/mL for the 24-hour treatment in the second experiment, the latter inducing a 58% decrease in the PD.

 

In the first experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels, either following the 3- or 24-hour treatments. Furthermore, no dose-response relationship in the frequency of micronucleated cells was demonstrated by the linear regression, whatever the treatment period.

Since none of the dose-levels analyzed in the first experiment reached the recommended level of cytotoxicity (i.e. 55±5% cytotoxicity), a second experiment was undertaken using a narrower range of dose-levels.

 

In the second experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels,either following the 3- or 24-hour treatments. Furthermore, no dose-response relationship in the frequency of micronucleated cells was evidenced, whatever the treatment period.

Despite the use of a narrower range of dose-levels, none of the dose-levels selected for the 3-hour treatment induced the recommended level of cytotoxicity. Considering the narrow dose-levels spacing used in this experiment and the negative results obtained in both independent experiments performed, the overall available results were considered as suitable to allow a reliable interpretation.

 

The overall results without S9 mix were considered to meet the criteria of a negative response.

 

Experiments with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:

- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL in the first experiment,

- 7.5, 15, 30, 40, 45, 50, 55 and 60 µg/mL in the second experiment.

 

No emulsion was observed in the culture medium at any dose-levels, at the end of the treatment periods.

 

In the first experiment, a slight to severe cytotoxicity was induced at dose-levels = 40 µg/mL, as shown by a 31 to 100% decrease in the PD.

In the second experiment, a moderate to severe cytotoxicity was induced at dose-levels = 30 µg/mL, as shown by a 45 to 92% decrease in the PD.

The dose-levels selected for micronucleus analysis were as follows:

- 10, 20 and 40 µg/mL in the first experiment, the latter inducing only a 31% decrease in the PD but higher dose-levels being too cytotoxic,

- 15, 30 and 40 µg/mL in the second experiment, the latter inducing a 52% decrease in the PD.

 

In the first experiment,a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 40 µg/mL (p < 0.05). However, in the absence of any dose-response relationship, the criteria of a positive response were only partially met. Thus, these results remained equivocal.

Since none of the dose-levels of the first experiment induced the recommended level of cytotoxicity (i.e. 55 ± 5% cytotoxicity), and since the results remained equivocal, a second experiment was undertaken using a narrower range of dose-levels.

 

In the second experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels and no dose-response relationship was evidenced. Since the slight increase observed in the first experiment was not reproduced in the second experiment, using a narrower range of dose-levels and reaching the recommended level of cytotoxicity, it was considered to be non biologically relevant. Thus the overall results with S9 mix met the criteria of a negative response.

 

Under the experimental conditions of the study, the test item 2-Hydroxy-3-phenoxypropyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

hprt locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver rat

Test concentrations with justification for top dose:

Preliminary solubility data indicated that 2-hydroxy-3-phenoxypropyl acrylate was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 210.1 mg/mL. The solubility limit in culture medium was in the range of 525.3 to 1051 µg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 3 hours after test article addition. A maximum concentration of 2000 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to the maximum recommended concentration according to current regulatory test guidelines (OECD, 2015). Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Range finder (+/-S9): 62.5-125-250-500-1000-2000 µg/ml
Mutation experiment (-S9): 2.5-5-10-15-20-22.5-25-27.5-30-32.5-35-40 µg/ml
Mutation experiment (+S9): 25-50-75-100-110-120-130-140-170-200-250 µg/ml

Vehicle / solvent:

DMSO
Test article stock solutions were prepared by formulating 2-hydroxy-3-phenoxypropyl acrylate under subdued lighting in DMSO, with the aid of vortex mixing (where required), to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2 hours of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control

- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.

Rationale for test conditions:

Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 62.5 to 2000 µg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations
(1000 and 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration (2000 µg/mL) in the absence of S-9 only.
The highest concentration to give =10% relative survival (RS) in the presence of S-9 was 125 µg/mL, which gave 21% RS. In the absence of S-9, complete toxicity (0% RS) was observed at all concentrations analysed (62.5 to 2000 µg/mL).
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (2000 µg/mL), compared to the concurrent vehicle controls

Mutation
In the Mutation Experiment twelve concentrations, ranging from 2.5 to 40 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 25 to 250 µg/mL, were tested in the presence of S-9. No precipitate was observed either
upon addition of the test article to the cultures or immediately following the 3 hour treatment incubation period. Seven days after treatment, the highest concentration (40 µg/mL) in the absence of S-9 and the highest three concentrations (170 to 250 µg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest concentration (2.5 µg/mL) in the absence of S-9 was not selected as there were sufficient concentrations to define an appropriate toxicity profile. All other concentrations were selected in the absence and presence of S-9, but only one culture was plated at 30 µg/mL in the absence of S-9 due to a technical error during treatment of the replicate culture which resulted in this culture being discarded. The highest concentrations analysed were 35 µg/mL in the absence of S-9 and 140 µg/mL in the presence of S-9, which gave 14% and 13% RS, respectively.
The acceptance criteria were met and the study was accepted as valid.
When tested up to toxic concentrations, no statistically significant increases in mean MF values were observed following treatment with 2-hydroxy-3-phenoxypropyl acrylate at any concentration tested in the absence and presence of S-9. A statistically significant linear trend (p=0.05) was observed in the presence of S-9 but, in the absence of any statistically significant increases in MF at any concentration analysed, this observation was considered not biologically relevant.

Conclusions:

It is concluded that 2-hydroxy-3-phenoxypropyl acrylate did not induce mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Executive summary:

2-hydroxy-3-phenoxypropyl acrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 62.5 to 2000 µg/mL (the maximum recommended concentration according to current regulatory test guidelines). The highest concentration to give =10% relative survival (RS) in the presence of S-9 was 125 µg/mL, which gave 21% RS. In the absence of S-9, complete toxicity (0% RS) was observed at all concentrations analysed (62.5 to 2000 µg/mL).

In the Mutation Experiment twelve concentrations, ranging from 2.5 to 40 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 25 to 250 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 35 µg/mL in the absence of S-9 and 140 µg/mL in the presence of S-9, which gave 14% and 13% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in mean

MF values were observed following treatment with 2-hydroxy-3-phenoxypropyl acrylate at any concentration tested in the absence and presence of S-9. A statistically significant linear trend (p=0.05) was observed in the presence of S-9 but, in the absence of any statistically significant increases in MF at any concentration analysed, this observation was considered not biologically relevant.

It is concluded that 2-hydroxy-3-phenoxypropyl acrylate did not induce mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2016 - 26 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

n/a

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found toxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
The selected dose-levels were:
- 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 102 strain in the first experiment,
- 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the TA 1535 and TA 100 strains in the first experiment,
- 2.3, 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the TA 1537 and TA 98 strains in the first experiment,
- 62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment,
- 125, 250, 500, 1000, 2000 and 5000 µg/plate for the TA 102 strain in the second experiment.

Experiments with S9 mix
The selected dose-levels were:
- 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the first experiment,
- 125, 250, 500, 1000, 2000 and 5000 µg/plate for the TA 102 strain in the first experiment and for the five strains in the second experiment,
- 250, 500, 1000, 1500, 2000, 3500 and 5000 µg/plate for the TA 1535 strain in the third experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: the test item was found to be soluble in DMSO at 100 mg/mL. Therefore, the highest recommended dose-level could be reached using a treatment volume of 50 µL/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second and third experiments with S9 mix were performed according to the pre-incubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn.

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, >= 5000 µg/plate in the TA98 strain without S9 mix
- Otrher confounding effects: none.

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 µL/plate, the highest recommended dose-level of 5000 µg/plate was achievable. Thus, the dose levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

A moderate precipitate was observed in the Petri plates when scoring the revertants at the highest dose level of 5000 µg/plate only in the TA 98 strain without S9 mix.

In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 100 µg/plate in the TA 98 strain, >= 1000 µg/plate in the TA 100 strain and >= 2500 µg/plate in the TA 102 strain.
In the presence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants) was noted at dose-levels >= 1000 µg/plate in the TA 98 strain and at the dose-level of 2500 µg/plate in the TA 100 strain. No noteworthy toxicity was noted in the TA 102 strain with S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document
- Negative (solvent/vehicle) historical control data: see attached document

ADDITIONAL INFORMATION ON TOXICITY:
- Measurement of toxicity used: The evaluation of the toxicity was performed on the basis of the observation of a decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Conclusions:

2-Hydroxy-3-phenoxypropyl acrylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in three independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Treatments were performed according to the direct plate incorporation method except for the second and third experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Since the test item was found toxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels, either with or without S9 mix.

Experiments without S9 mix

Selected dose-levels ranged from 2.3 to 5000 µg/plate.

In both experiments, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels = 1666.7 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains, and at the highest dose-level of 5000 µg/plate in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any strains, in either experiment. These results without S9 mix met the criteria of a negative response.

Experiments with S9 mix

Selected dose-levels ranged from 20.6 to 5000 µg/plate.

Using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity (decrease in the number of revertants) was noted at the highest tested dose-level of 5000 µg/plate in the TA 98 and TA 102 strains, and at dose-levels = 1666.7 µg/plate in the TA 1537 strain. No noteworthy toxicity was noted in the TA 1535 and TA 100 strain.

Using the pre-incubation method (i.e.second and third experiments), a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels = 2000 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains, and at the highest dose-level of 5000 µg/plate in the TA 102 strain.

An increase in the number of revertants was noted at 5000 µg/plate in the TA 1535 strain in the second experiment (pre-incubation method). This increase did not reach the positive threshold of 3-fold the vehicle control value but was very close (2.9-fold) and the mean number of revertants was above the vehicle historical range (38.3 versus [9-26]). However, there was no clear evidence of a dose-response relationship. Since the increase in the number of revertants observed in the second experiment was not reproduced in the third experiment (performed under the same experimental conditions) despite the use of a narrower range of dose-levels, it was considered not to be biologically relevant and the overall results met the criteria of a negative response.

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions, in either experiment. Thus, the overall results with S9 mix met the criteria of a negative response.

Conclusion

Under the experimental conditions of this study, 2-Hydroxy-3-phenoxypropyl acrylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria (Chevallier 2016) :

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in three independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Treatments were performed according to the direct plate incorporation method except for the second and third experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

In both experiments without S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels = 1666.7 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains, and at the highest dose-level of 5000 µg/plate in the TA 102 strain. The test item did not induce any noteworthy increase in the number of revertants, in any strains, in either experiment. These results without S9 mix met the criteria of a negative response.

Using the direct plate incorporation method (i.e.first experiment) with S9 mix, a moderate to strong toxicity (decrease in the number of revertants) was noted at the highest tested dose-level of 5000 µg/plate in the TA 98 and TA 102 strains, and at dose-levels=1666.7 µg/plate in the TA 1537 strain. No noteworthy toxicity was noted in the TA 1535 and TA 100 strain. Using the pre-incubation method (i.e.second and third experiments), a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels= 2000 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains, and at the highest dose-level of 5000 µg/plate in the TA 102 strain.

An increase in the number of revertants was noted at 5000 µg/plate in the TA 1535 strain in the second experiment (pre-incubation method). This increase did not reach the positive threshold of 3-fold the vehicle control value but was very close (2.9-fold) and the mean number of revertantswas abovethe vehicle historical range (38.3 versus [9-26]). However, there was no clear evidence of a dose-response relationship. Since the increase in the number of revertants observed in the second experiment was not reproduced in the third experiment (performed under the same experimental conditions) despite the use of a narrower range of dose-levels, it was considered not to be biologically relevant and the overall results met the criteria of a negative response. The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions, in either experiment. Thus, the overall results with S9 mix met the criteria of a negative response.

Under the experimental conditions of this study, 2-Hydroxy-3-phenoxypropyl acrylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.

In vitro cytogenicity / micronucleus in mammalian cells study (Chevallier 2016b):

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

After a preliminary toxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments with (3h treatment) and without (3hr and 24h treatment) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

In the first experiment without S9 mix, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels, either following the 3- or 24-hour treatments. Furthermore, no dose-response relationship in the frequency of micronucleated cells was demonstrated by the linear regression, whatever the treatment period. Since none of the dose-levels analyzed in the first experiment reached the recommended level of cytotoxicity (i.e.55±5% cytotoxicity), a second experiment was undertaken using a narrower range of dose-levels. In the second experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels,either following the 3- or 24-hour treatments. Furthermore, no dose-response relationship in the frequency of micronucleated cells was evidenced, whatever the treatment period. Despite the use of a narrower range of dose-levels, none of the dose-levels selected for the 3-hour treatment induced the recommended level of cytotoxicity. Considering the narrow dose-levels spacing used in this experiment and the negative results obtained in both independent experiments performed, the overall available results were considered as suitable to allow a reliable interpretation. The overall results without S9 mix were considered to meet the criteria of a negative response.

In the first experiment with S9 mix, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 40 µg/mL (p < 0.05). However, in the absence of any dose-response relationship, the criteria of a positive response were only partially met. Thus, these results remained equivocal. Since none of the dose-levels of the first experiment induced the recommended level of cytotoxicity (i.e. 55 ± 5% cytotoxicity), and since the results remained equivocal, a second experiment was undertaken using a narrower range of dose-levels. In the second experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels and no dose-response relationship was evidenced. Since the slight increase observed in the first experiment was not reproduced in the second experiment, using a narrower range of dose-levels and reaching the recommended level of cytotoxicity, it was considered to be non biologically relevant. Thus the overall results with S9 mix met the criteria of a negative response.

 Under the experimental conditions of the study, the test item 2-Hydroxy-3-phenoxypropyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

In vitro gene mutation in mammalian cells test (OECD 476, Lloyd 2017):

2-hydroxy-3-phenoxypropyl acrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The test article was formulated in anhydrous analytical dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment.

In the Mutation Experiment twelve concentrations, ranging from 2.5 to 40µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 25 to 250µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 35µg/mL in the absence of S-9 and 140µg/mL in the presence of S-9, which gave 14% and 13% RS, respectively.

When tested up to toxic concentrations, no statistically significant increases in mean MF values were observed following treatment with 2-hydroxy-3-phenoxypropyl acrylate at any concentration tested in the absence and presence of S-9. A statistically significant linear trend (p=0.05) was observed in the presence of S-9 but, in the absence of any statistically significant increases in MF at any concentration analysed, this observation was considered not biologically relevant.

It is concluded that 2-hydroxy-3-phenoxypropyl acrylate did not induce mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

 

Justification for classification or non-classification

Based on the three negative in vitro genotoxicity studies, no classification is required for genetic toxicity according to the Regulation EC n°1272/2008.